The present invention relates to a procedure for cloning genes that encode restriction endonucleases, and to the gene products synthesized by such clones. The procedure makes use of the fact that certain modification genes, when cloned into a new host and adequately expressed, enable the host to tolerate the presence of a variety of different restriction genes. These artificial combinations of modification and restriction genes so created can be as stable, biologically, as the naturally-occurring combinations of restriction and modification genes. More specifically, the present invention relates to the use of the M.HhaI modification gene to facilitate the cloning of the R.FspI restriction gene, and to the use of the M.FnuDI modification gene to facilitate the cloning of the R.HaeIII restriction gene. The present invention also relates to the restriction enzymes synthesized from genes cloned by this procedure, and, more specifically, to the R.FspI and R.HaeIII restriction enzymes.
Restriction endonucleases (`ENases`) are enzymes that occur mainly in bacteria; they function as simple immune systems that help cells to recover from infection by detrimental DNA molecules, such as viruses. Restriction endonucleases interact, with remarkable exactness, with particular sequences of nucleotides within DNA molecules -- `recognition sequences` -- and break apart the molecules in the vicinity of these sequences. Breakage disrupts the genes and leads, eventually, to the disintegration of the entire DNA molecule. The process is termed `restriction`.
Since bacteria contain DNA themselves, they have developed a way to protect their DNA from digestion by their own ENases. They do this by chemically altering the sequences in their DNA that would otherwise be recognized by the ENases. The ENases are unable to interact with, or cleave, the altered sequences. The process of alteration is termed `modification`. Modification is carried out by enzymes -- termed methyltransferases (MTases); it consists of the covalent attachment of a methyl group to one of the adenine or cytosine residues that occur in each strand of the recognition sequence. An ENase and a MTase, together, make up a restriction-modification (`R-M`) system. Some bacterial species possess only one R-M system; others have several R-M systems, each acting independently at a different DNA sequence. The biological interdependence of ENases and MTases has become so intertwined over time that the genes encoding these enzymes (`R` and `M` genes) have co-evolved, and occur together, for the most part, side-by-side, in bacterial chromosomes.
During the last fifteen years, restriction endonucleases, and to a lesser extent modification methyltransferases, have become useful laboratory reagents. The enzymes can be purified by conventional protein-purification techniques, and they can then be used to alter DNA molecules in the test tube. The altered DNA can be separated, analyzed, joined in new arrangements, and reintroduced into living cells. ENases, especially, provide the molecular biologist, the clinical researcher, and the forensic chemist, alike, with ways to dissect and to identify DNA molecules. Because of their usefulness, there is a strong incentive to develop strains of bacteria that yield high levels of restriction and modification enzymes. One way to achieve this is to transfer (`clone`) the genes for an R-M system into a new host cell, under circumstances where the genes can then be over-expressed. The present invention concerns a new approach to cloning restriction genes; the approach is exemplified by the cloning of the genes for the FspI and HaeIII systems into Escherichia coli.
Many hundreds of restriction-modification systems have been discovered (Roberts, R., Nucleic Acids Res. 16: r271-313 (1988)), and a substantial number -- approximately fifty -- have now been cloned, in full or in part (Wilson, G., Gene 74: 281-289 (1989)). To clone an R gene, it is usually also necessary to clone the companion M gene, so that the recipient cell has the wherewithal to protect its DNA from digestion by the new ENase. Cloning an R gene thus becomes, in practice, a matter of cloning both R and M genes. Two general selection procedures have been used to isolate such clones. The first step in both procedures is the construction of a DNA library, consisting of fragments of bacterial DNA ligated into a plasmid vector, and introduced into a new host, usually E. coli. The first procedure selects, in vivo, for clones that express the restricting phenotype: the library of cells is incubated with bacterial viruses, and cells that survive are collected. The second procedure selects, in vitro, for clones that express the modifying phenotype: the library of recombinant plasmids is incubated with a restriction enzyme that cleaves unmodified DNA, and surviving molecules are collected following transformation into a host cell.
The first procedure yields clones that carry the complete R-M system (Mann et al., Gene 3: 97-112 (1978); Kosykh et al., Mol. Gen. Genet. 178: 717-718 (1980); Walder et al., Proc. Natl. Acad. Sci. USA 78: 1503-1507 (1981); Bougueleret et al., Nucleic Acids Res. 12: 3659-3676 (1984)). In practice, however, this method is fraught with difficulties, and it is often not successful. The second procedure yields both clones that carry the complete R-M system (Kiss et al., Nucleic Acids Res. 13: 6403-6421 (1985); Slatko et al., Nucleic Acids Res. 15: 9781-9786 (1987); Karreman and De Waard, J. Bacteriol. 170: 2527-2532 (1988)), and clones in which only the M gene is intact (Szomolanyi et al., Gene 10: 219-225 (1980); Janulaitis et al., Gene 20: 197-204 (1983); Caserta et al., J. Biol. Chem. 262: 4770-4777 (1987); Chandrasegaran et al, Gene 70: 387-392 (1988)). The second procedure is not altogether free of problems (Lunnen et al., Gene 74: 25-32 (1988)), but it is successful on most occasions, and so it has become the method of choice for scientists working in this area.
In some instances, methylation by a particular MTase protects the DNA from cleavage not only by the companion ENase, but by related ENases, too. This happens when the recognition sequences of the ENases are the same as, or are subsets of, the recognition sequence of the MTase. For example, methylation by the M.FnuDI MTase (recognition sequence, GGCC) blocks cleavage by not only the R.FnuDI ENase (GGCC), but also by the isoschizomers, R.HaeIII, R.BsuRI, and R.NooPII (GGCC), and by R.AatI (AGGCCT), R.ApaI (GGGCCC), R.BalI (TGGCCA), R.EaeI (PyGGCCPu), R.EaoI (CGGCCG), and R.NotI (GCGGCCGC) (see McClelland and Nelson, Gene 74: 291-304 (1988) for a compilation of protective modifications). In such instances, once an initial MTase gene has been cloned, in order to clone the related restriction genes, it ought not to be necessary to clone their companion MTase genes as well, since the initial MTase can substitute for all of them. Thus, if the gene for the M.FnuDI MTase were cloned, to continue the example, cells containing that gene could be used as recipients for the cloning of the R.HaeIII, R.BsuRI, R NooPII, R.AatI, R.ApaI, R.BalI, R EaeI, R.EagI, and R.NotI ENase genes. The R genes could be cloned independently of their companion M genes; only the R genes would need to be cloned, and not the full R-M systems.
We refer to situations where one MTase can substitute for a number of other MTases as `heterospecific modification`. There are several circumstances under which one might want to take advantage of the phenomenon of heterospecific modification. During manipulation of an R-M system, to overexpress the R gene for example, it could be beneficial to remove the companion M gene to increase the accessibility of the R gene. This could be accomplished rather easily if a heterospecific M gene were present to compensate for the loss of the normal MTase. Also, since it is usually more straightforward to clone a single gene, rather than two genes, it should be easier to clone an R gene on its own, than it would be to clone a complete R-M system. Thus, provided that ENase-containing clones could be reliably selected or identified, isolating a new R gene clone under conditions of preexisting, heterospecific modification should be more efficient than attempting to isolate it together with its companion M gene. Certain R-M systems cannot, in fact, be cloned directly in a single step, anyhow (Howard et al., Nucleic Acids Res 14: 7939-7951 (1987); Brooks et al., Nucleic Acids Res. 17: 979-997 (1989)). In these instances, the protection afforded by the cloned companion M gene appears to be inadequate, and as a result, recipient cells succumb to digestion by the new ENase. It is only possible to clone these systems if the recipient cells are already modified prior to the arrival of the R gene. Premodification can, admittedly, be accomplished using a previously cloned companion MTase (Howard et al., 1987; Brooks, et al., 1989), but using a heterospecific MTase, instead, has several advantages, including broader applicability and the absence of DNA sequence homology. The absence of DNA sequence homology enables previously cloned parts of the R-M sytem to be used as probes to detect the presence of the R gene.
We describe here the application of heterospecific modification to the cloning into E. coli of two R-M systems: the FspI R-M system (TGCGCA) from Fischerella species, the cloning of which was facilitated by the the hhaIM MTase gene (GCGC); and the HaeIII R-M system (GGCC) from Haemophilus aegyptius, the cloning of which was facilitated by the fnuDIM gene (GGCC). The examples demonstrate the utility of heterospecific modification as a means for cloning ENase genes or complete R-M systems.